In conjunction with other members of the proposed marker network, we propose to (1) develop new methods for accurate quantitation of new markers; (2) evaluate marker performance, and (3) develop classification or stratification schema to improve management of bladder cancer patients. The long-term objective is to develop a matrix of markers that can detect frank and developing bladder cancer and predict biological potential based upon risk factor analysis or confidence parameters for the markers. Markers will be measured using quantitative fluorescence image analysis, which can measure multiple markers on single cells in relation to morphology. The markers to be measured are as follows. (1) Morphology, is a documented marker for undifferentiated tumors and serves as the reference point for integration of biochemical markers. (2) DNA ploidy, including stem cell ploidy and the presence of cells with >5C DNA, increase sensitivity of tumor detection by at least 20% over morphology. (3) A tumor related antigen, pl85, detected by the M344 antibody of Y. Fradet, has a strong selectivity for low-grade tumors. (4) F-actin, an early differentiation marker, has shown a strong correlation with bladder cancer risk (p < 0.001 in a stratified risk study). (5) Epidermal growth factor receptor (EGFR) and (6) the product of the neu oncogene, participate in growth control. (7) The product of the p53 tumor suppressor gene regulates division and differentiation. Methods to measure new probes will be developed using a generally applicable approach involving demonstration of specificity of probes, selectivity for cells of different types within the urothelium, optimization of probe and reagent concentrations, and standardization. Markers will be evaluated using a 3-stage approach with a pilot study, a stratified risk study and a prospective, longitudinal followup study. The pilot study will provide information to select the most appropriate method to quantify a marker and to design larger studies. The stratified risk study involves measuring the marker in a cross section of patients and controls with a graded risk of developing disease and with other urologic conditions to rapidly evaluate how markers fall within the time frame of development of cancer, determine the correlations among markers and their sensitivity and specificity to endpoints such as development of cancer, recurrence, progression, and response to therapy. These studies will be carried out both with exfoliated samples and with biopsy samples from tumor and field to identify the sequence of oncogenic changes. Markers will also be evaluated in longitudinal studies with continued followup with endpoints as above. Statistical techniques of survival analysis and risk factor analysis by logistic regression will be used to develop classification schemas that can improve patient management. As a member of the network, quantitative fluorescence image analysis will be applied to methods development using probes developed by ourselves or other members of the group, including analysis of samples from other investigators.